Skip to main content
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1979 Dec;76(12):6076–6080. doi: 10.1073/pnas.76.12.6076

Electron microscopic visualization of N-acetoxy-N-2-acetylaminofluorene binding sites in ColE1 DNA by means of specific antibodies.

G de Murcia, M C Lang, A M Freund, R P Fuchs, M P Duane, E Sage, M Leng
PMCID: PMC411805  PMID: 293703

Abstract

ColE1 DNA has been allowed to react in vitro with N-acetoxy-N-2-[14C]acetylaminofluorene in the range of 0-15 N-2-[14C]acetylaminofluorene residues bound per molecule of DNA, at the C8 of guanine residues. Purified rabbit antibodies to both N-2-(guanosine-8-yl)-acetylaminofluorene and native DNA that had reacted with N-acetoxy-N-2-acetylaminofluorene were shown by electron microscopy to recognize specifically the acetylaminofluorene-modified ColE1 DNA. The antibodies bound to DNA were visualized either per se or after reaction with goat anti-rabbit immunoglobulins coupled with ferritin. There was a linear relationship between the average number of antibodies bound per DNA molecule and the number of N-2-(deoxyguanosine-8yl)-acetylaminofluorene residues per DNA molecule. The slope of this straight line was equal to 0.4. Due to the bivalence of the immunoglobulins one would expect a value of 0.5; we actually observed an important fraction of the bound antibodies crosslinking two parts of the same (or of another) DNA molecule.

Full text

PDF
6078

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Clewell D. B. Nature of Col E 1 plasmid replication in Escherichia coli in the presence of the chloramphenicol. J Bacteriol. 1972 May;110(2):667–676. doi: 10.1128/jb.110.2.667-676.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Dubochet J., Ducommun M., Zollinger M., Kellenberger E. A new preparation method for dark-field electron microscopy of biomacromolecules. J Ultrastruct Res. 1971 Apr;35(1):147–167. doi: 10.1016/s0022-5320(71)80148-x. [DOI] [PubMed] [Google Scholar]
  3. Fuchs R. P. Arylamidation and arylation by the carcinogen N-2-fluorenylacetamide: a sensitive and rapid radiochemical assay. Anal Biochem. 1978 Dec;91(2):663–673. doi: 10.1016/0003-2697(78)90553-5. [DOI] [PubMed] [Google Scholar]
  4. Guigues M., Leng M. Reactivity of antibodies to guanosine modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene. Nucleic Acids Res. 1979 Feb;6(2):733–744. doi: 10.1093/nar/6.2.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Kriek E. Persistent binding of a new reaction product of the carcinogen N-hydroxy-N-2-acetylaminofluorene with guanine in rat liver DNA in vivo. Cancer Res. 1972 Oct;32(10):2042–2048. [PubMed] [Google Scholar]
  6. Lefèvre J. F., Fuchs R. P., Daune M. P. Comparative studies on the 7-iodo and 7-fluoro derivatives of N-acetoxy-N-2-acetylaminofluorene: binding sites on DNA and conformational change of modified deoxytrinucleotides. Biochemistry. 1978 Jun 27;17(13):2561–2567. doi: 10.1021/bi00606a016. [DOI] [PubMed] [Google Scholar]
  7. Leng M., Sage E., Fuchs R. P., Duane M. P. Antibodies to DNA modified by the carcinogen N-acetoxy-N-2-acetylaminofluorene. FEBS Lett. 1978 Aug 15;92(2):207–210. doi: 10.1016/0014-5793(78)80755-8. [DOI] [PubMed] [Google Scholar]
  8. Poirier M. C., Dubin M. A., Yuspa S. H. Formation and removal of specific acetylaminofluorene-DNA adducts in mouse and human cells measured by radioimmunoassay. Cancer Res. 1979 Apr;39(4):1377–1381. [PubMed] [Google Scholar]
  9. Poirier M. C., Yuspa S. H., Weinstein I. B., Blobstein S. Detection of carcinogen-DNA adducts by radiommunoassay. Nature. 1977 Nov 10;270(5633):186–188. doi: 10.1038/270186a0. [DOI] [PubMed] [Google Scholar]
  10. Sage E., Fuchs R. P., Leng M. Reactivity of the antibodies to DNA modified by the carcinogen N-acetoxy-N-acetyl-2-aminofluorene. Biochemistry. 1979 Apr 3;18(7):1328–1332. doi: 10.1021/bi00574a032. [DOI] [PubMed] [Google Scholar]
  11. Tomizawa J., Sakakibara Y., Kakefuda T. Replication of colicin E1 plasmid DNA in cell extracts. Origin and direction of replication. Proc Natl Acad Sci U S A. 1974 Jun;71(6):2260–2264. doi: 10.1073/pnas.71.6.2260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Voordouw G., Kam Z., Borochov N., Eisenberg H. Isolation and physical studies of the intact supercoiled, the open circular and the linear forms of ColE1-plasmid DNA. Biophys Chem. 1978 May;8(2):171–189. doi: 10.1016/0301-4622(78)80008-8. [DOI] [PubMed] [Google Scholar]
  13. Westra J. G., Kriek E., Hittenhausen H. Identification of the persistently bound form of the carcinogen N-acetyl-2-aminofluorene to rat liver DNA in vivo. Chem Biol Interact. 1976 Oct 2;15(2):149–164. doi: 10.1016/0009-2797(76)90160-5. [DOI] [PubMed] [Google Scholar]
  14. de Murcia G., Das G. C., Erard M., Daune M. Superstructure and CD spectrum as probes of chromatin integrity. Nucleic Acids Res. 1978 Feb;5(2):523–535. doi: 10.1093/nar/5.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES